Lipoproteins are the primary carriers of plasma cholesterol. They are micellar lipid-protein complexes (particles), having a surface film comprised of one or more proteins associated with polar lipids, that surrounds a cholesterol-containing core. Original classification of lipoproteins was based on their buoyant densities as measured by ultracentrifugation. Accordingly, four major density classes have been recognized, and subclasses within these exist.
The first class comprises the chylomicrons. They are the largest of the lipoproteins and are rich in triglycerides. The site of origin of the chylomicrons is the intestine. When chylomicrons are exposed to plasma or high density lipoprotein (HDL) in vitro, much of their complement of A apolipoproteins is lost, and C and E apolipoproteins are acquired. Chylomicrons also contain apolipoprotein B-48.
The second class of lipoproteins, the very low density lipoproteins (VLDL), is comprised of particles made in the liver and involved in triglyceride metabolism and transport from the liver. The apolipoproteins, apo B-100 and apo E, are the major constituents of the VLDL particle.
The third lipoprotein class, comprising low density lipoproteins (LDL), is a specific product of the catabolism of VLDL. The predominant apolipoprotein in LDL particles is apolipoprotein B-100, or apo B-100.
The fourth class, high density lipoprotein (HDL) contains two major apolipoproteins, apo A-I and apo A-II. One function of apo A-I is the activation of the plasma enzyme, lecithin-cholesterol acyltransferase, which is required for the esterification of free cholesterol on HDL for transport to the liver.
Plasma cholesterol is regulated in part by the LDL receptor and in part on the ability of the lipoprotein to carry cholesterol and bind the LDL receptor. Hofmann, et al., Science, 239:1277 (1988). This receptor is found on the surface of all cells where it mediates binding and internalization of the cholesterol-rich lipoproteins that provide membrane cholesterol; Brown, et al., J. Clin. Invest., 55:783 (1975); Goldstein, et al., Methods Enzymol., 98:241 (1983); and in some specialized cells substrate cholesterol for the production of bile acids or steroid hormones; Gwynne, et al., Endocr. Rev., 3:299 (1982). LDL receptor expression on cells is inversely regulated by the circulating concentration of LDL, i.e., the higher the circulating LDL the fewer LDL receptors on the cell surface Hofmann, et al., Science, 239:1277 (1988). LDL binding to the LDL receptor has been the focus of much research because of its importance in regulating the level of plasma cholesterol, which is considered a major risk factor for the development of coronary artery disease; Brown, et al., Scient. Amer., 251:58 (1984).
LDL binding to the LDL receptor is believed to be dependent upon the species of apolipoprotein present in the lipoprotein particle.
When compared to apolipoproteins A-1 or B, the relative concentration of apo E in plasma is low. However, apo E is instrumental in lipoprotein metabolism in several ways. Mahley, et al., J. Lipid Res., 25:1277-1294 (1984). It is a recognition site for several cellular lipoprotein receptors, including hepatocyte receptors for chylomicron and VLDL remnants [Hui, et al., J. Biol. Chem., 259:860-869 (1984); Shelburne, et al., J. Clin. Invest., 65:652-658 (1980)], receptors for LDL on hepatic and extrahepatic cells [Hui, et al., J. Biol. Chem., 256:5646-5655 (1981)] and receptors for VLDL on macrophages [Wang-Iverson et al., Biochem. Biophys. Res. Commun., 126:578-586 (1985)].
Lipoproteins are cleared from the plasma by binding to high-affinity receptors on liver cells and extrahepatic tissues such as the adrenal glands and ovaries. Kowal, R.C. et al., Proc. Natl. Acad. Sci. USA, 86:5810-5814, (1989). The LDL receptor specifically binds apo B and apo E-bearing lipoproteins. R. W. Mahley, Science, 240:622 (1988). Thus, apo E is of clinical importance for its role in binding LDL receptor and facilitating cholesterol clearance.
The LDL receptor-binding region of apo E has been mapped to an internal sequence including amino acid residues 140 to 160. Weisgraber, et al., J. Biol. Chem. 258:12348 (1983). Additionally, apo E binds the LDL receptor only when it is associated with a lipoprotein or phospholipid [Innerarity, et al., J. Biol. Chem., 254:4186 (1979)] and 4 apo E molecules bind the LDL receptor with an affinity that is 10 to 25-fold greater than the binding of a single molecule of apo B. R. W. Mahley, Science, 240:622 (1988).
Two distinct sets of receptors bind apo E-containing lipoproteins. The LDL receptor [Yamamoto et al., Cell, 39:27-38 (1984)], 70% of which is thought to be located on hepatic cells, binds VLDL and apo E-containing remnants of chylomicrons. The existence of a second set of LDL receptors, termed "remnant receptors", is inferred from studies showing that the plasma clearance of apo E-containing chylomicron remnants occurs at normal rates in animals with genetically defective LDL receptors.
Recently, an LDL receptor-related protein (LRP) has been found on the surface of hepatic cells. Herz et al., EMBO, 7:4119-4127 (1988). LRP shares cysteine-repeat sequences with LDL and has been shown to bind and mediate the extracellular clearance of apo E-containing lipoproteins. Kowal, R.C. et al. Proc. Natl. Acad. Sci. USA, 86:5810-5814, (1989).
Apo E-enriched lipoproteins have also been described to have a function in the immune system by inhibiting mitogen-or antigen-stimulated lymphocyte proliferation in vitro and in vivo. In the ovary, apo E inhibits androgen production by LH-stimulated cultured theca and interstitial cells; Dyer, et al., J. Biol. Chem., 263:10965 (1988).
In 1976 it was reported that a discrete lipoprotein fraction isolated from normal human plasma inhibited mitogen- and allogenic cell-stimulated human lymphocyte proliferation in vitro (Curtiss et al., J. Immunol., 116:1452, (1976)). This inhibitory plasma lipoprotein was termed LDL-In for Low Density Lipoprotein-Inhibitor because the active fraction is localized to a less dense subfraction of total LDL of density 1.006-1.063 g/ml. The characteristics of LDL-In-mediated inhibition in vitro are as follows: LDL-In has comparable inhibitory activity for phytohemagglutinin (PHA), pokeweed mitogen (PWM), and allogenic cell-stimulated human lymphocyte proliferation. The inhibitory activity of LDL-In is non-toxic and independent of mitogen concentration. Suppression by LDL-In is time dependent and approximately 18 hr of exposure of the lipoprotein to the lymphocytes before stimulation is required for maximum induction of a stable suppressed state. LDL-In does not inhibit .sup.3 H-thymidine uptake when it is added to the cultures 18-20 hr after stimulation, suggesting that this lipoprotein influences metabolic events associated with an early inductive phase of lymphocyte activation.
The immunosuppressive activity of LDL-In has been studied in a number of systems both in vitro and in vivo. To summarize, in vitro activities of LDL-In include suppression of: a) mitogen stimulated .sup.3 H-thymidine uptake, Curtiss et al., J. Immunol., 116:1452, (1976), b) allogenic cell-stimulated .sup.3 H-thymidine uptake (Curtiss et al., J. Immunol., 116:1452, (1976), Curtiss et al., J. Immunol., 118:1966, (1977)), c) the primary generation of cytotoxic T cells (Edgington et al., Regulatory Mechanisms in Lymphocyte Activation: Proceedinqs of the Eleventh Leukocyte Culture Conference., D.O. Lucas, ed. Academic Press, New York, pp. 736, (1977)), d) pokeweed mitogen stimulated immunoglobulin synthesis (Curtiss et al., J. Clin. Invest., 63:193, (1979)), and e) B-cell Epstein Barr Virus transformation (Chisari et al., J. Clin. Invest., 68:329, (1981)). In vivo LDL-In has been shown to inhibit: a) the primary humoral immune response to sheep red blood cells (Curtiss et al., J. Immunol., 118:648, (1977), DeHeer et al., Immunopharmacology, 2:9, (1979), Curtiss et al., Cell. Immunol., 49:1, (1980)), b) the primary generation of cytotoxic T-cells (Edgington et al., Regulatory Mechanisms in Lymphocyte Activation: Proceedings of the Eleventh Leukocyte Culture Conference., D.O. Lucas, ed. Academic Press, New York, pp. 736, (1977)), and c) immunologic attention of tumor growth (Edgington et al., Cancer Res., 41:3786, (1981), Edgington et al., Dietary Fats and Health., ACOS Monograph No. 10, Perkins and Visek, eds., pp. 901, (1981)).
The effects of lipoproteins on immune cell function in vivo are exceedingly complex. A major finding of the investigation of the physiologic implications of immunosuppression by LDL-In in vivo is that the observed functional outcome is strikingly dose dependent. This important concept is best illustrated by describing in more detail studies of the effects of LDL-In on the survival of experimental animals challenged with syngeneic tumors (Edgington et al., Cancer Res., 41:3786, (1981), Edgington et al., Dietary Fats and Health., ACOS Monograph No. 10, Perkins and Visek, eds., pp. 901, (1981)). Seemingly divergent effects of LDL-In are observed on the growth of the syngeneic SaD2 fibrosarcoma in DBA/2 mice. The growth of 1.times.10.sup.5 viable tumor cells in control mice without immunoprotection (i.e., 10-days prior immunization with 10.sup.-6 irradiated tumor cells) is detectable at 25 days and proceeds rapidly until death at about 43 days. In contrast, tumor growth is slower in immunoprotected mice. This tumor growth is characterized by a reduction in tumor mass of at least a half and no deaths by day 60. Intravenous administration of high doses of LDL-In 24 hr before immunoprotection with killed tumor cells abolishes the protective effect of immunization. This dose corresponds to a dose that is required to abolish both B-cell and T-cell effector cell functions. The administration of an intermediate dose of LDL-In before immunoprotection with the killed tumor cells has no discernable effect on the subsequent growth of the viable tumor cell challenge. In contrast, intravenous administration of even lower doses of LDL-In 24 hr before immunoprotection with killed tumor cells results in the enhancement of tumor rejection and host survival. This dose of LDL-In is concordant with the dose required for selective inhibition of suppressor cell function in vitro (Curtiss et al., J., Clin. Invest., 63:193, (1979)). Thus, depending upon the amount of immunoregulatory lipoprotein that a particular lymphocyte population is exposed to in vivo, very different functional outcomes will result.
Further substantiation that apo E and Apo B-containing lipoproteins are important regulators of lymphocyte function has come from studies of the inhibitory properties of fetal cord blood plasma lipoproteins (Curtiss et al., J. Immunol., 133:1379, (1984)). In these studies a direct correlation between apo E and inhibition was established.
Cardin et al., Biochem. Biophys. Res. Comm., 154:741-745 (1988) reported that a polypeptide portion of apo E having an amino acid residue sequence identical to that of apo E residues 141-155 inhibits lymphocyte proliferation when coupled to bovine serum albumin (BSA). However, conspicuously absent from the study of Cardin et al. was any control for cell viability allowing for a determination of whether or not the inhibition observed was due to cytotoxicity of the peptide-BSA conjugate.
By way of further background, Dyer et al., J. Biol. Chem., 263:10965-10973 (1988) reported that isolated lipid free rat Apo inhibits androgen production by the ovarian theca and interstitial cells induced by the gonadotropin, luteinizing hormone (LH).
More recently, Dyer et al, J. Biol. Chem., 266:15009-15015, (1991), have shown that only multimers, and not monomers, of the apo E polypeptide p141-155 exhibit the biological activity of apo E.